Glutathione Metabolism Contributes to the Induction of Trained Immunity

Abstract

The innate immune system displays heterologous memory characteristics, which are characterized by stronger responses to a secondary challenge. This phenomenon termed trained immunity relies on epigenetic and metabolic rewiring of innate immune cells. As reactive oxygen species (ROS) production has been associated with the trained immunity phenotype, we hypothesized that the increased ROS levels and the main intracellular redox molecule glutathione play a role in the induction of trained immunity. Here we show that pharmacological inhibition of ROS in an in vitro model of trained immunity did not influence cell responsiveness; the modulation of glutathione levels reduced pro-inflammatory cytokine production in human monocytes. Single nucleotide polymorphisms (SNPs) in genes involved in glutathione metabolism were found to be associated with changes in pro-inflammatory cytokine production capacity upon trained immunity. Also, plasma glutathione concentrations were positively associated with ex vivo IL-1β production, a biomarker of trained immunity, produced by monocytes of BCG-vaccinated individuals. In conclusion, glutathione metabolism is involved in the induction of trained immunity, and future studies are warranted to explore its functional consequences in human diseases.

Keywords: glutathione; innate immune memory; macrophages; metabolism; trained immunity.

Figure 1

Increased ROS levels of trained monocytes do not contribute to their enhanced pro-inflammatory cytokine production. (A) ROS levels at 2 h, 24 h, and 6 days after exposure of monocytes to β-glucan and BCG (n = 6/9 donors, pooled from 2/3 independent experiments. Fridman test Dunn’s multiple comparisons test). (B) Expression levels of genes involved in the antioxidant defense in monocytes exposed to β-glucan for 24 h in comparison to unstimulated cells. Expression presented as log₁₀(ratio). TNFα and IL-6 produced by β-glucan-trained macrophages after a 1 h pretreatment with (C) 0.5µM DPI (n = 6 donors, pooled from 2 independent experiments) and (D) 1 mM NAC, 50 µM AT or 0.5 µM AA (n = 6 donors, pooled from two independent experiments, two-way ANOVA, Sidak’s multiple comparisons test). (mean ± SEM, * p < 0.05).

Figure 2

Glutathione levels are modulated upon β-glucan exposure. (A) Reduced and oxidized glutathione intracellular levels in monocytes after 24 h exposure with 1 µg/mL β-glucan and 5 days after the resting period (n = 4 donors, pooled from two independent experiments). (B) Expression of genes involved in glutathione metabolism in monocytes exposed to β-glucan for 4 h and 24 h. Expression presented as log₁₀(ratio). (C) TNFα and IL-6 produced by β-glucan-trained macrophages after a 1 h pretreatment with 100 µM BSO (n = 9 donors, pooled from three independent experiments, * p < 0.05 two-way ANOVA, Sidak’s multiple comparisons test) (mean ± SEM).

Figure 3

Glutathione is associated with trained immunity features. Heatmap of the p-values of association (p < 9.99 × 10⁻³) between SNPs mapped to genes involved in glutathione metabolism and the magnitude of cytokine production capacity by monocytes trained in vitro with (A) β-glucan and BCG (n = 251 healthy volunteers for IL-6 and n = 238 healthy volunteers for TNFα) and (B) in vivo BCG training responses (n = 278 healthy volunteers). (C) Boxplot of the lowest p-value SNP per analysis (rs7768134, rs35568915). (D) Boxplots showing glutathione plasma levels (corrected for age and sex) stratified by genotype for rs2233294 (GPX3) and rs10136944 (GLRX5, n = 302). The p-value for each SNP is derived from linear regression model with the SNP of interest as independent variable and glutathione (corrected for age and sex) as the dependent variable. (E) Spearman correlations between circulating metabolites at baseline involved in glutathione metabolism versus fold changes of PBMC-derived S. aureus-induced IL-6, IL-1β, and TNFα responses measured at 14 or 90 days after BCG vaccination compared to baseline (n = 297 healthy volunteers). The table presents the Spearman’s rho for each correlation, and the correlations in red highlight significant positive correlations (* p < 0.05, ** p < 0.01). The correlation between glutathione concentration and the fold change of IL-1β production at 90 days compared to day 0 is given as an example in a scatter plot.